Summary/Abstract Sepsis is characterized by dysregulated host responses. Thrombosis is a common complication of sepsis and may contribute to prolonged hospitalization, organ failure, and death. These adverse outcomes are more common in older septic patients. Platelets and their precursor cell, the megakaryocyte (MK), mediate the pathobiology of sepsis but the molecular underpinnings of these dysregulated functions in sepsis and aging remain poorly understood. Our proposal, ?Novel Regulatory Mechanisms of Inflammation-Induced Thrombosis? will use in vitro, in vivo, and clinical studies to determine how interferons (IFNs) generated during sepsis alter gene expression and functional responses in MKs and platelets. Our preliminary data demonstrate that the transcriptional and translational landscape of MKs and platelets is transformed during sepsis. We have identified a previously unrecognized protein in human platelets, interferon-induced transmembrane protein 3 (IFITM3). Platelet and MK IFITM3 is robustly induced in septic patients and models of IFN-stimulation. Induction of IFITM3 on MKs and platelets controls fibrinogen endocytosis, aggregation, and thrombosis. We hypothesize that IFNs released during sepsis trigger MK and platelet IFITM3 expression, inducing hyperreactivity and thrombosis. Aim 1 will use complementary pharmacological and genetic toolsets to determine how inflammatory agonists alter the regulation of IFITM3 in MKs and platelets. We will also establish if inflammation-induced platelet IFITM3 controls clathrin-mediated endocytosis of fibrinogen and other proteins and determine the necessity of activated integrin ?IIb?3 for this pathway. Aim 2 will delineate the functional effects of MK and platelet IFITM3. Using younger and older mice where Ifitms have been globally silenced, as well as mice where Ifitm3 is silenced only in MKs and platelets, we will determine whether IFITM3 upregulates fibrinogen endocytosis and platelet aggregation. We will also employ two established murine models of thrombosis, mimicking the thrombotic complications in sepsis, to determine if the genetic ablation of Ifitm3 protects against thrombosis in the presence of inflammatory agonists. Aim 3 will determine whether platelet IFITM3 regulates thrombosis in sepsis. We will determine if fibrinogen endocytosis and platelet aggregation are enhanced in platelets isolated from older and younger septic patients, where IFITM3 is increased. In parallel, we will identify if increased platelet IFITM3 in murine sepsis (e.g. cecal ligation and puncture, CLP) promotes thrombosis and whether there are aging-related changes in IFITM3 regulation and thrombosis. These studies are translational and innovative. They will determine for the first time whether IFNs present in the septic milieu reprogram MKs and developing platelets, contributing to thrombotic complications of sepsis. The impact of these studies will also extend beyond sepsis to other age-related human diseases associated with thrombosis where IFNs are dysregulated and where targeting IFNs may have therapeutic benefit.